Subunit vaccine of Pasteurella multocida in veterinary uses

ABSTRACT

The present invention declaims the use of  Pasteurella  lipoprotein E (PlpE) as a subunit vaccine and the use of vaccines containing PlpE to protect animals from diseases caused by  P. multocida . The results of vaccination and challenge experiments showed that mice and chickens immunized with PlpE were completely protected animals from challenge infection with 10 1 -10 3  LD 50  of  P. multocida  and no adverse effect was observed.

FIELD OF THE INVENTION

The present invention relates to the use of Pasteurella lipoprotein E(PlpE) in a subunit vaccine, and the use of vaccines containing PlpE toprotect animals from diseases caused by P. multocida.

BACKGROUND OF THE INVENTION

Pasterurella multocida is classified into family Pasteurellaceae, genusPasteurella, and multocida means “causing many kinds of animal disease”in Latin. In the past, the taxonomic situation of Pasteurellaceae wasambiguous and arguable. However, according to the sequence analysis of16s-RNA, Pasteurellaceae is classified into a gamma subgroup of purplebacteria, which is Gram-negative facultative anaerobe, and homologous toEnterobacteriaaceae. Pasteurella multocida is an important pathogen ofdomestic animals and an opportunistic pathogen of humans. Humaninfections with P. multocida largely arise from the bite of an infectedcarnivore, but other types of infections are occasionally reported(Hubbert W T, et al., Am J Public Health 1970; 60:1109-17). P. multocidahas wide host range, and is the causative agent of fowl cholera indomestic birds, haemorrhagic septicaemia in cattle or sheep, andatrophic rhinitis in pigs (Hunt M L, et al., Vet Microbiol 2000,72:3-25). Most P. multocida strains are highly pathogenic to murine andrabbits, which can result in acute septic symptoms at an infection doseof 1-10 CFU.

In domestic birds, P. multocida causes acute septic disorders in turkey,chicken, duck, and goose, which may lead to a great loss in economy. Ofthe five capsular serotypes (A, B, D, E and F) and 16 LPS serotypes,fowl cholera is mainly caused by serotypes A:1, A:3 and A:4 (Glisson JR. In: Saif Y M, editor. Diseases of poultry. Iowa State UniversityPress, Ames, Iowa, 2003:657-90). Although both live-attenuated vaccinesand bacterins are available, outbreaks of fowl cholera continue tooccur. Live-attenuated vaccines have the disadvantage of reversion tovirulence, while bacterins do not protect hosts against heterologouschallenge (Bierer B W, Derieux W T, Poult Sci 1972; 51:408-16; andRebers P A, Heddleston K L, Avian Dis 1977; 21:50-56). Thesedisadvantages call for the development of a new type of vaccine for P.multocida.

In a previous report, a lipoprotein, designated Pasteurella lipoproteinE (PlpE), from Mannheimia haemolytica (formerly known as Pasteurellahaemolytica), was found to be highly immunogenic in cattle (Confer A W,et al., Vaccine 2003; 21:2821-9). PlpE is a lipid-modified,surface-exposed outer membrane protein that is important incomplement-mediated killing of M. haemolytica (Pandher K, et al., InfectImmun 1998; 66:5613-9). Addition of recombinant PlpE to the commercialM. haemolytica vaccine markedly enhanced the vaccine-induced resistanceagainst experimental challenge with serotypes 1 and 6. Abioinformatics-based sequence search showed that a gene annotated PlpEis present in the published genome sequence of P. multocida strain pm-70(serotype A:3) (May B J, et al., Proc Natl Acad Sci USA 2001;98:3460-5). This gene has the potential to encode a lipoprotein of 335amino acids that has 24.3% sequence identity with PlpE of M. haemolyticaand 19.1% identity with OmlA of A. pleuropneumoniae. It has not yet beendetermined whether the PlpE of P. multocida could serve as a vaccineantigen by using mice and/or chicken as animal models.

Therefore, the present invention first finds out a new use ofPasteurella lipoprotein E (PlpE) in controlling infective diseasescaused by P. multocida, or related disorders thereof, and furtherdevelops a subunit vaccine for protecting animal from diseases caused byP. multocida. The subunit vaccine of present invention is characterizedby using recombinant PlpE protein as active antigen, which has noadverse side effect of forming fibrosarcoma. Additionally, the presentsubunit vaccine of P. multocida can provide a predominant protectiveeffect against the challenge of homologous and/or heterologous serotypesin immunized animals over traditional inactivated or live-attenuatedvaccines.

SUMMARY OF THE INVENTION

In one aspect, the present invention provides a subunit vaccine forprotecting animal from diseases caused by P. multocida, which ischaracterized by comprising Pasteurella lipoprotein E (PlpE) as antigen,and a veterinary acceptable adjuvant. In one embodiment, the Pasteurellalipoprotein E is a protein having the amino acid sequence as listed inSEQ ID EF219452-EF219457 (SEQ ID NO:2, 4, 6, 8, 10 and 12), or an aminoacid sequence with similarity of more than 90% to the amino acidsequence as listed in SEQ ID EF219452-EF219457 (SEQ ID NO:2, 4, 6, 8, 10and 12). In another embodiment, the disease caused by P. multocida isfowl cholera in domestic birds, haemorrhagic septicaemia in cattle, oratrophic rhinitis in pigs.

In another aspect, the present invention provides a use of Pasteurellalipoprotein E in controlling infective diseases caused by P. multocida,or related disorders thereof. The disease caused by P. multocidainfection may be fowl cholera in domestic birds, haemorrhagicsepticaemia in cattle, or atrophic rhinitis in pigs. In other animals,such as mice, rabbits, cats, or dogs, P. multocida infection may causehaemorrhagic septicaemia.

BRIEF DESCRIPTION OF DRAWINGS

FIG. 1. Expression and purification of recombinant PlpE (r-PlpE) andrecombinant PlpB (r-PlpB) in E. coli. Note that r-PlpB was used as thecontrol antigen in this report. (A) Coomassie blue-stained SDS-PAGE ofrecombinant proteins from crude extract or purified samples. Lane Mrepresents the molecular mass markers. The lanes marked control containcrude extract of E. coli that harbored no recombinant plasmid. (B)Immunoblot of duplicated gel probed with mouse anti-hexa-histidinemonoclonal antibody. The bands corresponding to r-PlpB, r-PlpE, andtheir processed products are indicated by arrows.

DETAILED DESCRIPTION OF THE INVENTION

The present invention will be further defined by reference to thefollowing examples, which are set forth to assist in understanding theinvention and should not be construed as specifically limiting theinvention. Therefore, any modification or derivative made withoutdeparting from the spirit of this invention will be considered to fallwithin the scope of the invention.

EXAMPLE Example 1 Preparation of Recombinant Lipoprotein E (r-PlpE) ofP. multocida in E. coli

A. Bacterial Strains and Genomic DNA Extraction of P. multocida

P. multocida standard strains X-73 (A:1), P-1059 (ATCC 15742) (A:3), andP-1662 (A:4) were grown at 37° C. in BRAIN-HEART INFUSION (BHI) broth(Difco Laboratories, MI, USA) for 18-24 hours. Bacterial genomic DNA wasisolated using the DNEASY® TISSUE KIT (QIAGEN, Hilden, Germany).

B. Genetic Cloning and Expression Vector Construction for RecombinantLipoprotein E (r-PlpE)

One set of primers, P1 and P2, was used to amplify the PlpE gene from P.multocida strain X-73. The amplified genes were then used for expressingrecombinant PlpE (r-PlpE) in E. coli. These primers containedrestriction enzyme (NcoI or XhoI) cutting sites at their 5′-ends(underlined sequences), followed by sequences specific to PlpE. The PCRproduct was cloned into the expression vector pET28a according to themanufacturer's instructions (Novagen, Inc. Madison, Wis.) to obtain aplasmid designated as pX73-PlpE. The identity of the insert in pET28awas verified by DNA sequence analysis. The amino sequence of r-PlpEprotein is described in SEQ ID: 2.

C. Expression and Purification of Recombinant Lipoprotein E (r-PlpE)

Recombinant plasmid pX73-PlpE obtained in section B was transformed intoE. coli strain BL21 (DE3) and recombinant protein was purified by nickelchromatography as previously described (Chang P C, et al., 2002, AvianDis 46:570-80). In brief, E. coli strain BL21 (DE3) harboring therecombinant plasmid was cultured in LB medium at 37° C. until absorbanceat 600 nm reached 0.6. Isopropylthio-β-D-thiogalactose (IPTG) was addedto a final concentration of 0.4 mM, and the culture was grown foranother 3 hrs. Cells were pelleted by centrifugation at 3000×g for 20min, and resuspended in 2 ml of binding buffer (20 mM pH 7.9 Tris, 5 mMimidazole, 500 mM NaCl). The suspension was sonicated and centrifuged at12,000×g for 40 min. The supernatant was collected and loaded into acolumn containing 2.5 ml of “His-bind” resin (Novagen). The column waswashed with 25 ml of binding buffer and 15 ml of washing buffer (20 mMpH 7.9 Tris, 50 mM imidazole, 500 mM NaCl) to remove the unboundproteins. The bound protein was eluted with 15 ml of eluting buffer (20mM pH 7.9 Tris, 250 mM imidazole, 500 mM NaCl), only the first 3 ml ofthe elute was collected. Protein concentration was determined using a“Protein Assay” kit (BIO-RAD, Hercules, Calif., USA).

The expression product and purity of the recombinant protein wasobserved by SDS-PAGE and Western blotting analysis, respectively. Theresults were showed in FIG. 1. The r-PlpB in FIG. 1 was a noneffectivesubunit vaccine used as a control in the experiments. The plpB and plpEgenes were cloned from P. multocida strain X-73 (serotype A:1) and thenexpressed in E. coli as recombinant proteins. The recombinant PlpB(r-PlpB) and PlpE (r-PlpE) contained a hexa-histidine-tag attached attheir carboxyl termini.

As showed in FIG. 1, the calculated molecular masses of r-PlpB andr-PlpE were 31.5 and 38.7 kDa, respectively. Both r-PlpB and r-PlpEcontained a signal peptide of 20 amino acid residues at their aminotermini, and after cleavage of the signal peptide, the matured r-PlpBand r-PlpE had molecular masses of 29.3 and 36.3 kDa, respectively. Asshown in FIG. 1A, r-PlpB and r-PlpE, with the expected molecular masses,were highly expressed in E. coli and were purified using nickelchromatography (FIG. 1A).

Western blot analyses using anti-hexa-histidine monoclonal antibodyshowed that this monoclonal antibody reacted with r-PlpB and r-PlpE(FIG. 1B); moreover, both r-PlpB and r-PlpE produced two bands on theblot, the major band having the molecular mass corresponding to thefull-length r-PlpB or PlpE (31.5 or 38.7 kDa), whereas the minor bandhad the molecular mass of the mature form (29.3 or 36.3 kDa) (FIG. 1B).This result suggests that some processing of r-PlpB and r-PlpE occurredin E. coli. The bands corresponding to r-PlpB, r-PlpE and theirprocessed products are indicated by arrows.

Example 2 Evaluation of Protective Effects of r-PlpE Subunit Vaccine inBALB/c Mice Model

Three experiments were conducted in BALB/c mice. In experiments 1 and 2,groups of 6-week-old mice were immunized subcutaneously with 10micrograms of purified r-PlpB or r-PlpE in aluminum hydroxide adjuvant(Sigma-Aldrich Co., MO, USA), either alone or together with a bacterincomposed of 1.25×10⁷ or 2.5×10⁷ CFU of formalin-inactivated P. multocidaX-73 (A:1). Two weeks after immunization, mice were challenged withsubcutaneous injection of 10-20 LD₅₀ of strain X-73. In experiment 3,mice were immunized as described for experiments 1 and 2. Two weeksafter immunization, mice were challenged with subcutaneous injection of10 LD₅₀ of strains P-1059 (A:3) or P-1662 (A:4), or strain T2A5 (whichis a designated challenge strain used in drug inspection in Taiwan). Allmice challenged were observed for 10 days and their survival rates wererecorded. The results are summarized in Table 1. For statisticalanalysis, the survival rates were compared by Chi-squared tests usingSAS® software (SAS Institute, Inc., Cary, N.C., USA). The mean times todeath were compared using the GLM procedure in the same software.Differences were considered significant when p<0.05.

TABLE 1 Results of immunization and challenge tests in BALB/c mice.Challenge strain Immunized with¹ and dose² % survival³ Exp. 1 X-73 (A:1)Control 30 CFU 0 (0/6)^(a) r-PlpB (10 microgram) 30 CFU 0 (0/10)^(a)r-PlpE (10 microgram) 30 CFU 100 (10/10)^(b) Inactivated X-73 (2.0 × 10⁸CFU) 30 CFU 100 (6/6)^(b) Inactivated X-73 (1.25 × 10⁷ CFU) 30 CFU 17(1/6)^(a) Inactivated X-73 (1.25 × 10⁷ CFU) + 30 CFU 30 (3/10)^(a)r-PlpB (10 microgram) Inactivated X-73 (1.25 × 10⁷ CFU) + 30 CFU 10(10/10)^(b) r-PlpE (10 microgram) Exp. 2 X-73 (A:1) Control 60 CFU 0(0/6)^(a) r-PlpB (10 microgram) 60 CFU 10 (1/10)^(a) r-PlpE (10microgram) 60 CFU 80 (8/10)^(bc) Inactivated X-73 (2.5 × 10⁷ CFU) 60 CFU50 (3/6)^(ab) Inactivated X-73 (2.5 × 10⁷ CFU) + 60 CFU 40 (4/10)^(ab)r-PlpB (10 microgram) Inactivated X-73 (2.5 × 10⁷ CFU) + 60 CFU 90(9/10)^(c) r-PlpE (10 microgram) Exp. 3 P-1059 (A:3) Control 35 CFU 0(0/5)^(a) r-PlpE (10 microgram) 35 CFU 100 (10/10)^(b) P-1662 (A:4)Control 30 CFU 0 (0/5)^(a) r-PlpE (10 microgram) 30 CFU 100 (10/10)^(b)T2A5 (A:1) Control 3 CFU 0 (0/5)^(a) r-PlpE (10 microgram) 3 CFU 80(8/10)^(b) ¹Mice in the control group were not immunized. ²The LD₅₀ ofstrains X-73 and P1662 in mice was <3 CFU, and that of P-1059 was 3.5CFU. ³Different alphabetical characters indicate significant difference(p < 0.05) between groups.

In experiment 1, mice immunized with 10 microgram of purified r-PlpEwere completely protected (100% survival) (Table 1, experiment 1). Incontrast, mice immunized with 10 microgram of purified r-PlpB were notprotected (0% survival) against challenge infection with 30 CFU (>10LD₅₀) of X-73 (serotype A:1). Mice immunized with a bacterin composed of2×10⁸ CFU of formalin-inactivated X-73 were completely protected (100%survival), whereas those immunized with a bacterin composed of a lowerdose (1.25×10⁷ CFU) of X-73 were not protected (17% survival) (Table 1,experiment 1). To investigate whether r-PlpB or r-PlpE could enhance theprotective efficacy of the bacterin, mice were immunized with a bacterincomposed of 1.25×10⁷ CFU of X-73 supplemented with 10 microgram r-PlpBor r-PlpE. The results showed that r-PlpB did not significantly enhancethe protective efficacy of the bacterin (30% survival, p>0.05) whereasr-PlpE did (100% survival, p<0.05) (Table 1, experiment 1).

In experiment 2, the challenge dose of X-73 was increased to 60 CFU (>20LD₅₀) and a bacterin composed of 2.5×10⁷ CFU of X-73 was used. Theresults showed that mice immunized with 10 microgram of r-PlpB were notprotected (10% survival) whereas those with 10 microgram of r-PlpE weresignificantly protected (80% survival, p<0.05) (Table 1, experiment 2).Mice immunized with a bacterin composed of 2.5×10⁷ CFU of X-73 weremoderately protected (50% survival). Mice immunized with the samebacterin supplemented with r-PlpB showed a survival rate of 40%, whichwas similar to that with the bacterin alone. In contrast, mice immunizedwith the bacterin supplemented with r-PlpE showed a survival rate of90%, which was significantly higher than that with the bacterin alone(p<0.05) (Table 1, experiment 2).

In experiment 3, strains P-1059 (serotype A:3) and P-1662 (serotype A:4)were used as the challenge strains. The results showed that miceimmunized with 10 microgram of r-PlpE were completely protected againstchallenge infection with 10 LD₅₀ of P-1059 or >10 LD₅₀ of P-1662 (Table1, experiment 3). This result showed that r-PlpE, which was derived fromX-73 (serotype A:1), conferred cross protection on mice againstchallenge with strains of serotypes A:3 and A:4. Additionally, miceimmunized with 10 microgram of r-PlpE showed a survival rate of 90% whenchallenge with strain T2A5 (A:1), which was up to the proof inspectionstandard (survival rate of 60%) (p<0.05) (Table 1, experiment 3).

Example 3 Evaluation of Protective Effects of r-PlpE Subunit Vaccine inSPF Chicken Model

Three experiments in SPF chickens were conducted. In experiment 1,groups of 3-week-old SPF chickens were immunized subcutaneously with 100micrograms of purified r-PlpB or r-PlpE in complete Freund's adjuvant(Sigma-Aldrich). Three weeks after the primary immunization, a boosterimmunization was conducted, and three weeks after booster immunization,chickens were challenged with intramuscular injection of 3.6×10³ CFU ofstrain X-73 or 5.5×10⁸ CFU of strain P-1662. In experiments 2 and 3,chickens were immunized subcutaneously twice with 125 micrograms of acrude extract of r-PlpE in a double emulsion adjuvant with a 3-weekinterval between immunizations. The crude extract was prepared bysonicating the pellet of E. coli that expressed r-PlpE. The doubleemulsion adjuvant contained Marcol 52 oil (63%), Arlacel A (7%), andTween 80 (1.5%). Three weeks after booster immunization, chickens werechallenged by intramuscular injection of 3.6×10³−3.6×10⁶ CFU of strainX-73 or 5.5×10⁷−5.5×10⁹ CFU of strain P-1662. All chickens challengedwere monitored for 10 days and the survival rates were recorded. Theresults are summarized in Table 2. For statistical analysis, thesurvival rates were compared by Chi-squared tests using SAS® software(SAS Institute Inc., Cary, N.C., USA). The mean times to death werecompared using the GLM procedure in the same software. Differences wereconsidered significant when p<0.05.

TABLE 2 Results of immunization and challenge tests in SPF chickensChallenge strain Mean time to Immunized with ¹ and dose ² % survival ³death (days) ³ Exp. 1 X-73 (A:1) Control 3.6 × 10³ CFU 30 (3/10)^(a)3.3^(a) Purified r-PlpB (100 microgram) 3.6 × 10³ CFU 50 (5/10)^(a)4.2^(a) Purified r-PlpE (100 microgram) 3.6 × 10³ CFU 100 (10/10)^(b) NAP-1662 (A:4) Control 5.5 × 10⁸ CFU 13 (1/8)^(a) 4.1^(a) Purified r-PlpB(100 microgram) 5.5 × 10⁸ CFU 13 (1/8)^(a) 5.4^(a) Purified r-PlpE (100microgram) 5.5 × 10⁸ CFU 63 (5/8)^(b) 5.7^(a) Exp. 2 X-73 (A:1) Control3.6 × 10³ CFU 25 (2/8)^(a) 2.7^(a) 3.6 × 10⁴ CFU 0 (0/8)^(a) 1.9^(a) 3.6× 10⁵ CFU 0 (0/8)^(a) 2.0^(a) Crude extract r-PlpE 3.6 × 10³ CFU 100(8/8)^(b) NA (125 microgram) 3.6 × 10⁴ CFU 75 (6/8)^(b) 7.0^(b) 3.6 ×10⁵ CFU 75 (6/8)^(b) 5.0^(b) 3.6 × 10⁶ CFU 88 (7/8)^(b) 4.0^(b) Exp. 3P-1662 (A:4) Control 5.5 × 10⁷ CFU 25 (2/8)^(a) 5.7^(a) 5.5 × 10⁸ CFU 13(1/8)^(a) 4.1^(a) 5.5 × 10⁹ CFU 0 (0/8)^(b) 2.9^(a) Crude extract r-PlpE5.5 × 10⁷ CFU 50 (4/8)^(a) 4.8^(a) (125 microgram) 5.5 × 10⁸ CFU 50(4/8)^(a) 4.8^(a) 5.5 × 10⁹ CFU 50 (4/8)^(a) 5.3^(a) ¹ Immunization wasconducted twice with a 3-week interval. Chickens in the control groupwere not immunized. ^(2, 3) Different alphabetical characters indicatesignificant difference (p < 0.05) between immunization and controlgroups challenged with the same dose of X-73 or P-1662.

In experiment 1, chickens immunized twice with 100 microgram of purifiedr-PlpB showed a survival rate of 50% against challenge with X-73, butthis survival rate was not significantly higher than that of the controlgroup (30% survival, p>0.05). In contrast, chickens immunized twice with100 microgram of purified r-PlpE showed a survival rate of 100%, whichwas significantly higher than that of the control group (p<0.05) (Table2. experiment 1). This result suggests that r-PlpE but not r-PlpBconferred protection on chickens. A similar conclusion was reached whenstrain P-1662 was used as the challenge strain (Table 2, experiment 1).

In experiments 2 and 3, a crude extract of r-PlpB and r-PlpE (FIG. 1A),instead of the purified one, was used as the antigen. Moreover, a doubleemulsion adjuvant, instead of Freund's complete adjuvant, was used asthe emulsifying agent. These modifications were carried out to reducethe cost and labor required for preparation and administration of theantigen. The results showed that chickens immunized twice with 125microgram of crude extract of r-PlpE had a survival rate of 75-100%against challenge with 3.6×10³−3.6×10⁶ CFU of strain X-73. These rateswere significantly higher than those of the control group (p<0.05)(Table 2, experiment 2). Moreover, the mean time to death of chickensimmunized with r-PlpE was significantly longer than that of the controlgroup (p<0.05) (Table 2, experiment 2).

In experiment 3, P-1662 was used as the challenge strain. The resultsshowed that chickens immunized with 125 microgram of crude extract ofr-PlpE had a survival rate of 50% against challenge with 5.5×10⁷−5.5×10⁹CFU of strain P-1662. These rates were not significantly higher thanthose of the control groups (p>0.05), except when the challenge dose ofP-1662 was 5.5×10⁹ (p<0.05) (Table 2, experiment 3). The mean times todeath of immunized chickens were not significantly longer than those ofthe control groups (p>0.05) (Table 2, experiment 3).

Example 4 Nucleotide Sequences of plpE from Reference Strains of P.multocida

Two primers, P3 and P4, were used to amplify the PlpE genes fromdifferent reference strains of P. multocida, X-73 (A:1), pm-70 (A:3),P-470 (A:3), P-61 (D:3), P-1059 (A:3), P-1662 (A:4), and ATCC 12948(D:11). The two primers were designed on the basis of the publishedgenome sequence of P. multocida strain pm-70. P3 and P4 amplified the1.0 kb DNA fragment containing the PlpE gene. The sequences of primersP3 and P4 were as follows. P3: 5′-ATG AAA CAA ATC GTT TTA AA-3′ (SEQ IDNO:13), and P4: 5′-TTA TTG TGC TTG GTG ACT TT-3′ (SEQ ID NO:14). The PCRproducts were purified with a QIAQUICK® GEL EXTRACTION KIT (QIAGEN) andsequenced from both directions using a BIGDYE® TERMINATOR CYCLESEQUENCING KIT (Applied Biosystems, Foster City, Calif.) in an automaticsequencer (ABI-3730XL DNA ANALYZER®, Applied Biosystems). Sequences werecompiled using the SEQMAN® program in the LASERGENE® package (DNASTARInc. Madison, Wis., USA). Open reading frames prediction and antigenicindex assay were performed using the GENEQUEST and PROTEAN programs fromthe same package. Nucleotide and protein sequences were searched forhomology in GenBank using the BLAST program provided by NCBI, USA.

The nucleotide sequences of the PlpE gene determined in this study areavailable in GenBank under the accession numbers EF219452-EF219457(corresponding to the SEQ ID NO:1, 3, 5, 7, 9, and 11) in the appendingsequence listing). All these PlpE genes were found to contain an openreading frame of 1008-1019 nt, encoding a PlpE protein of 37.4-37.7 kDa.Pair-wise sequence comparison showed that these PlpE proteins had90.8-100% sequence identity with each other, suggesting that PlpE mightserve as a cross-protective antigen. This is the first report of arecombinant P. multocida antigen that confers cross protection onanimals. Therefore, a protein having the amino acid sequence as listedin SEQ ID NO:2, 4, 6, 8, 10, and 12, or an amino acid sequence withsimilarity of more than 90% to the amino acid sequence as listed in SEQID NO:2, 4, 6, 8, 10, and 12, is considered to exhibit highly similarprotective effects, and contemplates to be included in the subunitvaccine of the present invention.

The above examples are given by way of illustration only, and should notbe construed as specifically limiting the scope of present invention.Any variation of the invention described and claimed herein, includingthe substitution of all equivalents, which would be within the purviewof those skilled in the art, is to be considered to fall within thescope of the invention incorporated herein.

The strain E. coli BL21 (DE3) containing the recombinant vector X73-plpEof the invention was deposited with the Agricultural Research ServiceCulture Collection (NARRL), on Feb. 29, 2008, as Deposit No. NARRLB-50117.

1. A subunit vaccine composition for controlling animal diseases causedby P. multocida, the subunit vaccine composition comprising recombinantPasteurella lipoprotein E (PlpE) as an antigen, and a veterinaryacceptable adjuvant, wherein the Pasteurella lipoprotein E is a proteinhaving the amino acid sequence identified by SEQ ID NO:2.
 2. A subunitvaccine composition of claim 1, wherein the animal disease caused by P.multocida is haemorrhagic septicaemia and pneumonic pasteurellosis incattle.
 3. A subunit vaccine composition of claim 1, wherein the animaldisease caused by P. multocida is fowl cholera.
 4. A subunit vaccinecomposition of claim 1, wherein the animal disease caused by P.multocida is atrophic rhinitis and pneumonic pasteurellosis in pigs.